Poulopoulos, A. | bioRxiv Science

bioRxiv2020

NPRL3: Direct Effects on Human Phenotypic Variability, mTOR Signaling, Subcellular mTOR Localization, Cortical Lamination, and Seizure Susceptibility

Nitrogen Permease Regulator Like 3 (NPRL3) variants are associated with malformations of cortical development (MCD) and epilepsy. We report a large (n=133) founder NPRL3 (c.349delG, p.Glu117LysFS) pedigree dating to 1727, with heterogeneous epilepsy and MCD phenotypes. Whole exome analysis in individuals with and without seizures in this cohort did not identify a genetic modifier to explain the variability in seizure phenotype. Then as a strategy to investigate the developmental effects of NPRL3 loss in human brain, we show that CRISPR/Cas9 Nprl3 knockout (KO) in Neuro2a cells (N2aC) in vitro causes mechanistic target of rapamycin (mTOR) pathway hyperactivation, cell soma enlargement, and excessive cellular aggregation. Amino acid starvation caused mTOR inhibition and cytoplasmic mTOR localization in wildtype cells, whereas following Nprl3 KO, mTOR remained inappropriately localized on the lysosome and activated, evidenced by persistent ribosomal S6 and 4E-BP1 phosphorylation, demonstrating that Nprl3 loss decouples mTOR activation from metabolic state. Nprl3 KO by in utero electroporation in fetal (E14) mouse cortex resulted in mTOR-dependent cortical dyslamination with ectopic neurons in subcortical white matter. EEG recordings of these mice showed hyperexcitability in the electroporated hemisphere. NPRL3 variants are linked to a highly variable clinical phenotype likely as a consequence of mTOR-dependent effects on cell structure, cortical development, and network organization.

neuroscience↗

bioRxiv2020

Cas9 fusions for precision in vivo editing

Cas9 targets genomic loci with high specificity. When used for knockin, however, Cas9 often leads to unintended on-target knockout rather than intended edits. This imprecision is a barrier for direct in vivo editing where clonal selection is not feasible. Here we demonstrate a high-throughput workflow to ratiometrically assess on-target efficiency and precision of editing outcomes. Using this workflow, we screened combinations of donor DNA and Cas9 variants, as well as fusions to DNA repair proteins. This yielded novel high-performance double-strand break repair editing agents and combinatorial optimizations with orders-of-magnitude increases in knockin precision. Cas9-RC, a novel Cas9 fusion to eRad18 and CtIP, increased knockin performance over 3-fold in vitro and in vivo in the developing mouse brain. Continued comparative assessment of existing and novel editing agents with this ratiometric framework of efficiency and precision will further the development of direct in vivo knockin and future genetic therapies.

synthetic biology↗